Alleviating product inhibition of Trichoderma reesei cellulase complex with a product-activated mushroom endoglucanase.

Product inhibition of cellulase is a challenging issue in industrial processes. Here, we introduced a product-activated mushroom cellulase, PaCel3A from Polyporus arcularius, into Trichoderma reesei. The filter paper activity, carboxymethyl cellulase activity, and saccharification efficiency (substrate: pretreated rice straw, PRS) of transformants increased significantly with this enzyme (by 18.4-26.8%, 13.8-22.8%, and 17.0%, respectively). A mutant of PaCel3A, PaCel3AM, obtained based on B-factor analysis, saturated mutagenesis, and residual activity assay, showed improved thermostability. The PRS saccharification efficiency using the cellulase complex from T. reesei transformants overexpressing pacel3am increased by 56.4%-63.0%. In addition, the T. reesei cellulase complex obtained by adding the purified recombinant PaCel3AM from T. reesei (rCel3aM-tr) to hydrolyze PRS resulted in increased reducing sugar yields at all sampling points, outperforming the cellulase complexes without rCel3aM-tr. These results suggest that introducing product-activated cellulase genes is a simple and feasible method to alleviate the product inhibition of cellulase.

Chilling stress reduced protein translation by the ubiquitination of ribosomal proteins in Volvariella volvacea.

In Volvariella volvacea, an important edible mushroom species, cryogenic autolysis is a typical part of abnormal metabolism; however, the underlying mechanisms remain unclear. Ubiquitylome analysis revealed that chilling stress (CS) affected protein translation and degradation by ubiquitination. Comparative proteomics analysis showed that CS downregulated protein expression in V. volvacea V23 instead of VH3 (improved chilling stress resistance strain). The integrative ubiquitylome, proteomics, and transcriptome analyses indicated that CS reduced protein translation by the ubiquitination of ribosomal proteins. An activity assay of the 20S proteasome showed that CS decreased the degradation efficiency of the ubiquitin-proteasome system. UBEV2, one type of ubiquitin-conjugating enzyme E2 (UBE2) in V. volvacea, was upregulated after cold stress treatment using western blot analysis. GST pull-down experiments of UBEV2 provided evidence that CS affected protein translation by the ubiquitination of ribosomal proteins. Co-IP experiments confirmed that UBEV2 bound to the ubiquitinated SSB2, a ribosome-associated molecular chaperone. An anti-freezing experiment demonstrated that the UBE2 inhibitor could improve the cold stress resistance of V. volvacea. Our observations revealed that CS triggered ubiquitination-mediated autolysis associated with a decrease in protein translation and highlighted the mechanistic role of UBEV2 in facilitating cryogenic autolysis in V. volvacea. SIGNIFICANCE: Volvariella volvacea, the edible straw mushroom, is a highly nutritious food source widely cultivated on a commercial scale in tropical and subtropical regions. The challenges associated with the cryogenic autolysis preservation of V. volvacea have limited its marketability. This issue of cryogenic autolysis is both an interesting scientific problem to solve and a practical economic matter. Integrative ubiquitylome, proteomics, and transcriptome analyses, together with GST pulldown and Co-IP experiments, indicated that chilling stress reduced protein translation by the ubiquitination of ribosomal proteins in V. volvacea. This study significantly contributes to our understanding of ubiquitination-mediated autolysis associated with a decrease in protein translation in V. volvacea. Our data highlight the mechanistic role of UBEV2 in facilitating the cryogenic autolysis of V. volvacea. We provided a new idea for the preservation of V. volvacea by inhibiting UBEV2 to increase its marketability.